We collected oral and/or rectal swabs and serum from dogs and cats living in homes with SARS-CoV-2-PCR-positive persons for SARS-CoV-2 PCR and serology testing. Pre-COVID-19 serum samples from dogs and cats were used as negative controls, and samples were tested in duplicate at different timepoints. Raw ELISA results scrutinized relative to known negative samples suggested that cut-offs for IgG seropositivity may require adjustment relative to previously proposed values, while proposed cut-offs for IgM require more extensive validation. A small number of pet dogs  and one cat  were positive for SARS-CoV-2 RNA, and 28.6 and 37.5% of cats and dogs were positive for anti-SARS-CoV-2 IgG, respectively. Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in the coronavirus disease 2019 (COVID-19) pandemic, which has an ongoing, significant impact on human health and wellbeing. Regionally, at the time and place where this study was conducted, cases rose drastically from 101 reported cases per week in March 2020 to 40,407 cases per week in December 2020 . Since tPrevious studies have reported that companion animals develop an immune response to SARS-CoV-2 . HoweverHere we applied new methods for interpreting SARS-CoV-2 serology results in pet dogs and cats from SARS-CoV-2-PCR-positive homes through replicate measurements in target populations and pre-COVID-19 control samples. With these outcomes, we assessed the relationship between owner-answered questionnaires on pet behavioral and demographic data with seropositivity. Additionally, we measured SARS-CoV-2 RNA in samples using established methodology.The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.This was a prospective, observational descriptive study. Households were recruited from an approximately 100-mile radius surrounding Durham, NC, from March 2020–March 2021. A written consent form was obtained for each study participant and pet enrolled in the study.This study was a collaboration between Duke University and North Carolina State University, and was part of an ongoing study known as the Molecular and Epidemiological Surveillance in Suspected Infection (MESSI) study. Human sample collection was approved by the Duke Institutional Review Board (IRB) under the MESSI protocol number Pro00100241. Animal collection protocols were reviewed and approved by the Duke Institutional Animal Care and Use Committee (IACUC) before approaching an owner for permission to collect samples under the Molecular and Epidemiological Surveillance in Suspected Infection in House Pets (MESSI-HP) protocol number A079-20-03. The MESSI study team consisted of lab members with expertise in epidemiological field research and human sample collection. The MESSI-HP study team included one member of the MESSI field team with animal handling experience, one veterinary technician, and one veterinarian, who was present for house pet serum sample and oral and/or rectal swab collection. Inclusion and exclusion criteria are listed in The MESSI study team collected serial biological samples  of humans throughout the course of their illness (days 0 (day of enrollment in MESSI study), 1, 3, 7, 14, 21, 28, 45, 60, 120).Biological samples  were collected from house pets (dogs and cats) on visit days 0, 1, 3, 7, 14, 21, 28, 45, 60, 120, and/or 180. Some sample collection timepoints were omitted due to challenges with pet compliance or insufficient team members present who were comfortable assisting in animal sample collection. Oral and/or rectal swabs from exposed house pets were collected from days 0–60. Serum samples from exposed house pets were collected on day 28 or later to allow for seroconversion and to limit the number of people exposed to an actively infected or clinically ill human.Oral swabs were collected by placing a sterile polyester-tipped applicator (manufacturer Puritan 25-806-1P) into the house pet’s mouth and rubbing the cheeks, gums, and tongue. Rectal swabs were collected by placing an applicator into the rectum and swabbing gently in a circular motion. Polyester-tipped applicators were immediately placed in viral transport media (VSM01) from Dasky  in a disposable tube for transport and storage. Serum samples were collected using standard venipuncture and processing for transport and storage.Serum samples collected from house pets of SARS-CoV-2-positive households are referenced as “exposed serum samples” throughout.Forty-three (24 canine and 19 feline) pre-2019 serum samples submitted to the North Carolina State University’s Vector-Borne Disease and Diagnostic Laboratory (NCSU-VBDDL) for vector-borne disease testing prior to the emergence of SARS-CoV-2  were used as single-masked SARS-CoV-2-negative serological controls. Serum samples were frozen and stored at −20 °C until use in this study. Throughout the paper, these are referenced as “pre-2019 serological controls”.Oral and rectal swab samples collected from house pets from SARS-CoV-2-positive households are referenced as “exposed PCR samples” throughout the paper.Ten oral/rectal swab samples from six house pets (three dogs and three cats) from SARS-CoV-2-PCR-negative homes were included in PCR testing. These samples came from house pets belonging to owners who were enrolled in the MESSI study based on the aforementioned inclusion/exclusion criteria but who were found to be negative for SARS-CoV-2 by PCR. Throughout the paper, these are referenced as “unexposed PCR controls”.Diagnostic testing for SARS-CoV-2). Automated QIAsymphony  two-step RT-PCR and World Health Organization E_Sabeco primer-probe sets  were used (10) from 800 µL of sample were considered positive based on a threshold determined by the manufacturer to indicate a positive test. To improve the accuracy in reporting positive tests, samples with LOQ ≥ 62 RNA copies/mL were subsequently run on the COBAS 6800 system, a qPCR test approved for emergency use by the FDA in 2021 to detect SARS-CoV-2 RNA in swab samples    . This tezerland) . Sampleszerland) .Enzyme-linked immunosorbent assay (ELISA) was used for the detection of house pet IgG and IgM to SARS-CoV-2 in the pre-2019 serological controls and exposed serum samples as previously described by . AdsorptFrom the MESSI-HP serum samples and the pre-2019 serological controls, serum with a volume of at least 0.5 mL was divided into two aliquots, and the ELISA was performed on each aliquot. Staff performing the ELISA were masked to the identity of samples and group  for the second round of serology. These samples were used to calculate a false positive rate from initial reporting of “positive” and “negative”.Three questionnaires were provided to the primary owner. Each questionnaire was in paper copy and filled out either by a primary owner , or by one of the study team members while verbally asking each question to the owner. When possible, the same primary owner provided answers at each visit. Questionnaire 1  collectep-values and odds ratios evaluating associations between 14 behavioral factors and seropositivity. Adjusted p-values were calculated using Bonferroni’s correction to account for multiple comparisons. The full reproducible code is available at https://github.com/t-gin/SARS-CoV-2_in_housepets.Statistical tests were conducted using R 4.1.1 . DescripExposed PCR samples were collected from day 0 through day 60, with most swabs taken at day 0  and day 28 . Thirty-five house pets (9 cats and 26 dogs) had oral or rectal swabs taken at more than one time point.n = 13 samples), 60 (n = 2 samples), 120 (n = 22 samples), and 180 (n = 2 samples).Thirty-nine exposed serum samples were submitted for antibody testing. One sample was removed due to suspected erroneous results, as indicated by an outlier during visualization , resultiThe anti-SARS-CoV-2 IgG and IgM O.D.s from replicate testing for the exposed serum samples were evaluated alongside the O.D.s for the pre-2019 serological controls. Four of the 38 exposed serum samples (one from dogs and three from cats) did not have adequate volume for two aliquots; these were excluded from the analysis to establish an O.D. cut-off. To visually assess for quantitative consistency, O.D.s for round 1 and round 2 serology from the exposed serum samples were plotted in With the new cutoff points chosen, the false positive rate for IgG seropositivity in our pre-2019 serological controls was 0/24 (0%) in dogs and 1/19 (5.3%) in cats. A false positive rate for IgM was not calculated, as samples did not cluster such that a distinct cutoff point could be established. This contrasts with the ELISA cut-off procedure described earlier in our methods section, as well as in a previous manuscript, which resulted in a false positive rate for IgG seropositivity in the pre-2019 serological controls of 3/24 (12.5%) in dogs and 6/19 (31.6%) in cats.  Based onBased on the seropositive thresholds established with the pre-2019 serological controls, 9/24 (37.5%) dogs and 4/14 (28.6%) cats were IgG-positive. A breakdown of positive samples by timepoint includes 7/13 (53.8%) day 28 samples, 0/2 (0%) day 60 samples, 6/22 (27.3%) day 120 samples, and 1/2 (50%) day 180 samples. A summary of results from seropositive animals is provided in Three of 64 (4.8%) animals from 2/32 (6.3%) SARS-CoV-2-positive households were deemed SARS-CoV-2 positive based on a positive result from the QuantStudio 3 RT-PCR and the COBAS 6800 analyzer. Of the 38 animals with serology results, 36  had a complete Questionnaire 2, and 38  had a complete Questionnaire 3 from the time of sample collection. Questionnaires were analyzed with serum data but not PCR data due to the large number of missing questionnaires from animals with PCR but not serology and very few PCR-positive animals.p = 0.04) or furniture (p = 0.048). A negative association was identified between dogs within IgG-positive ELISA and owners reporting that dogs were known to lick plates in the dishwasher (p = 0.04). None of these associations were found to be significant following Bonferroni’s correction, which is represented by the “adjusted p-value” in Fourteen exposures were tested for a relationship with IgG seropositivity in dogs and cats. Associations and calculated statistics are reported in p = 0.40). Three out of nine (33.3%) IgG-positive dogs were reported to have symptoms at one or more timepoints during sample collection. A table of the testing timeline and results for each of these dogs is included in supplementary materials  but within the range of PCR-positive animals from studies of a similar design. No stastitically significant associations were identified between questionnaires related to owner-reported animal symptomology or behavior and IgG-seropositivity after correcting for multiple hypotheses.A key finding in this study is that in the SARS-CoV-2 ELISA performed here, IgG in dogs had the clearest and most consistent results compared to IgM in dogs, and IgG and IgM in cats, based on distinct clustering of results from exposed serum samples between two runs and when compared to pre-2019 serological controls . There wInterestingly, two of our PCR-positive animals were IgG-negative at day 60. However, this may represent contamination of SARS-CoV-2 RNA from the household family members or environment rather than true infection. Regarding the PCR-positive animals, a Ct < 37 for targets 1 and 2 was used as the cutoff for determining a positive COBAS 6800 result. Newer research since the time of our sample testing suggests that a Ct < 33 for both targets is more specific for SARS-CoV-2 . The facCompared to other North American studies of a similar design, our IgG seroprevalence falls on the upper range for dogs (11–41%) and the lower end of the range for cats (21–51%) . The varWe attempted to ascertain relationships between seropositive pets and characteristics of the pet-owner relationship through questionnaires. Prior to correcting for multiple comparisons, we found significant positive relationships between dogs allowed on the bed or furniture and IgG-seropositivity, as well as a significant negative relationship between dogs that lick plates in the dishwasher and IgG-seropositivity. None of these behaviors were found to be significant after Bonferroni’s correction. However, the first two associations may have merit in terms of a relationship, as it would make sense that dogs that share furniture and bedding are more likely to come into contact with the virus. With that in mind, the positive association between dogs being allowed on the couch or bed and having a higher likelihood of seropositivity aligns with findings in a previous study and thus may warrant further exploration.. We did Our study was strengthened through the use of negative controls and repeat testing of the same samples in the same lab using the same methodology. However, our study is not without limitations. The small sample size may limited our power to identify relationships between behavioral factors and seropositivity. This is highlighted by the fact that, prior to Bonferroni’s correction, licking plates in the dishwasher had a negative relationship with SARS-CoV-2 seropositivity in dogs, and contact with furniture had a positive relationship with seropositivity. Although not demonstrated in this study, it is possible that such relationships could be validated through larger sample sizes.In addition to sample size, our study would have benefited from using multiple different types of control samples, including pre-2019 negative controls for PCR, known positive serum samples for calculating a false negative rate, and swab and serum samples positive for other types of coronaviruses to assess for cross-reactivity. Unfortunately, we were unable to locate such sample types and thus attempted to increase the strength of determining whether an animal was positive for viral genes through the use of two testing modalities (RNA and the COBAS 6800 system). Moreover, we did not collect data on whether dogs and cats had been previously vaccinated for other coronaviruses which theoretically could cross-react with SARS-CoV-2 serology. Additionally, we were limited by the availability of owners and willingness to participate at any given time, which resulted in a variable sampling timeline. Lastly, the results of this study reflect early patterns of the SARS-CoV-2 (prior to any variants) and may not represent the role of companion animals with the current SARS-CoV-2 variants. Nevertheless, the results emphasize the importance of using robust control methods when validating and reporting test results.versus the much higher false positive rates reported from the initial ELISA procedure.This study did not generate strong evidence to support or negate animals as a significant reservoir for transmission of SARS-CoV-2, although that was not a specific aim. However, we brought into question the validity of “positive” and “negative” results reported for SARS-CoV-2 ELISAs that have not been evaluated with a large number of negative controls, specifically IgM in dogs and cats and IgG in cats. This is demonstrated with the false positive rates calculated based on the cutoff points we established With respect to serology, virus neutralization (V.N.) could have contributed to the strength of our findings, as it is often considered a gold standard for specific antibody activity. Still, the lack of V.N. does not necessarily take away from the main finding that SARS-CoV-2 ELISA should be thoroughly investigated prior to reporting. Virus neutralization could be considered for parallel sampling of pets when investigating shared infections in the future.The main goal of our study was to evaluate SARS-CoV-2 PCR and serology in a population of dogs and cats living in households with SARS-CoV-2-PCR-positive humans, as well as provide robust analytical methods for validating these results. Few (3/64) animals were PCR-positive for SARS-CoV-2 RNA, despite living in homes with known SARS-CoV-2-PCR-positive humans, and had high Ct values and no documentation of seroreactivity, supporting the possibility that these PCR positives were all compatible with environmental contamination. A larger number of animals (13/38), including 38% of dogs (9/24) and 29% of cats (4/14) were seropositive for IgG, as determined through robust ELISA validation methods. At the day 28 sampling timepoint, over 50% of sampled pets were IgG seropositive (7/13).10.7717/peerj.16310/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.16310/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.16310/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.16310/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.16310/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.16310/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.16310/supp-7Supplemental Information 7Click here for additional data file.


N-stearoylethanolamine (NSE) on diverse cancer cell lines, including multidrug-resistant models. No antioxidant or cytoprotective effects of NSE were found when it was applied together with doxorubicin. A complex of NSE with the polymeric carrier poly(5-(tert-butylperoxy)-5-methyl-1-hexen-3-yn-co-glycidyl methacrylate)-graft-PEG was synthesized. Co-immobilization of NSE and doxorubicin on this carrier led to a 2-10-fold enhancement of the anticancer activity, particularly, against drug-resistant cells overexpressing ABCC1 and ABCB1. This effect might be caused by accelerated nuclear accumulation of doxorubicin in cancer cells, which led to the activation of the caspase cascade, revealed by Western blot analysis. The NSE-containing polymeric carrier was also able to significantly enhance the therapeutic activity of doxorubicin in mice with implanted NK/Ly lymphoma or L1210 leukemia, leading to the complete eradication of these malignancies. Simultaneously, loading to the carrier prevented doxorubicin-induced elevation of AST and ALT as well as leukopenia in healthy Balb/c mice. Thus, a unique bi-functionality of the novel pharmaceutical formulation of NSE was revealed. It enhanced doxorubicin-induced apoptosis in cancer cells in vitro and promoted its anticancer activity against lymphoma and leukemia models in vivo. Simultaneously, it was very well tolerated preventing frequently observed doxorubicin-associated adverse effects.This study reports a dose-dependent pro-apoptotic action of synthetic cannabimimetic  The low selectivity of action of anticancer drugs and their numerous negative side effects on normal tissues of cancer patients belong to the most significant drawbacks in chemotherapy ,2. AnothIn order to reduce the general toxicity of anticancer drugs, novel approaches were developed for anticancer chemotherapy selectively targeting certain molecules or metabolic pathways overexpressed or hyperactivated in cancer cells . HoweverImmobilization of highly toxic anticancer drugs on bio-functionalized delivery platforms can reduce the effective drug concentration as well as increase tumor specificity . Here esThe use of nanoparticles as highly effective, non-toxic, and biodegradable platforms for drug delivery to target cells has become widespread. For this purpose, liposomes, carbon, silicon, iron, and gold nanomaterials were proposed. However, they all have certain disadvantages preventing their broad use in clinical practice . Here, wN-acylethanolamines (NAE) were chosen. These biologically active lipids belong to a group of cannabinoid-like compounds, that are produced by neurons and some other cells of the body in response to diverse physiological agents or stress [Immobilization of the Dx on the polymeric carrier reduced its effective dose, however, it did not influence its general toxicity . Therefor stress .N-stearoylethanolamine (NSE), a representative of the NAE group, inhibited the growth of Lewis lung carcinoma in mice and reduced the extent of metastatic lung lesions in tumor-bearing animals [Previously, we have established the neuroprotective effect of the synthetic NAE at chronic morphine addiction as well as their cardioprotective effect in an in vivo model of experimental heart ischemia ,10. More animals . AlthougMaterials. The peroxide monomer 5-tert-butylperoxy-5-methyl-1-hexen-3-yne (VEP) was synthesized, as described [420 = 0.867 g/mL, nd20= 1.4482. Glycidyl methacrylate (GMA)  was purified by distillation in vacuum. The poly(ethylene glycol) methyl ether , azobis(isobutyronitrile) (AIBN) and boron trifluoride ethyl etherate were obtained from Merck  and used without additional purification. The solvents ethyl acetate, dioxane, chloroform (Chl), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich and used after distillation.escribed . After vSynthesis. The comb-like copolymer poly(VEP-co-GMA)-graft-mPEG was synthesized in the following order [3⋅OEt2), the obtained polymer was purified by re-precipitation from solution in dioxane into hexane [ng order . Initialo hexane . Then, tn) and weight average (Mw) molecular weights were determined using gel-penetration chromatography on a Styragel HR1 (THF) column . Tetrahydrofuran (THF) was used as eluent and polystyrene of known molecular weight were used as standard. The flow rate was 0.5 mL/min. The assumed structure of PEG-containing comb-like polymer is presented in the scheme (The composition of poly(VEP-co-GMA)-graft-mPEG was determined using elemental  and funce scheme .Preparing the waterborne system without Dx and NSETo prepare the PC-based micelles in aqueous solutions, 0.09 g of PC was dissolved in 0.9 mL DMSO. This PC solution was added to 8.0 mL 0.9% NaCl. Then, the solution was stirred for 0.5 h and sonicated for 10 s.Preparing the waterborne system without Dx but with NSETo prepare the PC- and NSE-based micelles in aqueous solutions, 0.09 g of PC was dissolved in a 0.45 mL mixture of DMSO: Chl (the ratio of DMSO: Chl = 3:1 mass), 0.03 g of the NSE was also dissolved in a 0.45 mL mixture of DMSO: Chl. PC and NSE solutions were mixed. This solution of PC and NSE was added to 8.0 mL 0.9% NaCl. Then, the solution was stirred for 0.5 h and sonicated for 10 s. The solution was stirred while heating at 40 °C for another 1 h to remove traces of Chl.Preparing the waterborne delivery system with Dx but without NSEWater dispersions of the micelle of PC with Dx were obtained by dropping the solution of Dx and PC in DMSO into water, in the following order: 0.09 g of PC was dissolved in 0.25 mL of DMSO and 0.003 g of Dx was dissolved in 0.20 mL of DMSO. The solutions of PC and Dx were mixed, added dropwise to 8.0 mL 0.9% NaCl and sonicated for 10 s.Preparing the waterborne delivery system with Dx and NSEThe water dispersion of the PC- and NSE-based micelle complex loaded with Dx was obtained by dropping the solution of Dx, PC, and NSE into water, in the following order. First, 0.09 g PC, 0.003 g NSE, and 0.003 g Dx were dissolved in 0.2 mL, 0.15 mL, and 0.10 mL of a DMSO: Chl mixture, respectively. Then, the solutions were mixed, added to 8.0 mL 0.9% NaCl and sonicated for 10 s. Subsequently, the solution was stirred, while heating at 40 °C for another 1 h to remove traces of Chl. Subsequently, the aqueous dispersion was again treated by ultrasound for 10 s.The sizes of the polymer micelles of PC and of the complexes of PC with Dx and NSE were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS  and DynaPro NanoStar  and by photon correlation spectra using the NIBS (non-invasive back scatter) at 25 °C. The samples for DLS measurements were prepared as described above, and if necessary were diluted with bi-distilled water, pH 6.5–7.0. Three–five measurements were made for every sample .Zeta potential measurements were carried out with a Zetasizer Nano ZS at a fixed temperature of 25 °C. In total six measurements were performed at each water dispersion and the average value was calculated. The zeta potential data were reproducible within a precision of better than 5%. Studies of Dx release from drug delivery systemDx release from micellar complexes was studied in model experiment with saline using Pur-A-Lyzer™ Maxi Dialysis Kit . An amount of 3 mL of solution of pure Dx or micellar complexes Dx-PC and Dx-PC-NSE were placed into Maxi dialysis tubes (pore size 3.5 kDa MWCO) and plunged in 30 mL of saline (0.9% sodium chloride). All flasks were incubated at 37 °C under mild agitation, and samples were collected at different time intervals. Release of Dx from the micellar complexes was analyzed by measuring UV spectra of dialysate at 220 nm on Nanodrop ND-1000 , and plotted as a function of time to determine drug release kinetics. All experiments were performed at least three times with good reproducibility.Dx hydrochloride was obtained from Pfizer . NSE was synthesized at the Department of Lipids Biochemistry, Palladin Institute of Biochemistry  using the condensation of ethanolamine with stearic acid . Purity The sample of NSE for gas chromatography was prepared as follows: 0.1 mg of NSE, 1 mL of benzene, and 1 mL of 3 M HCl in methanol were mixed in a glass ampoule. The ampoule was sealed and heated in a water bath at 100 °C for 1.5 h. After this procedure, ampoule was cooled and opened. The solution was removed, filled into a sample tube, and mixed with 5 mL of hexane. The upper fraction of the solution was collected and transferred into a glass flask. The glass flask with the NSE sample was then dried up using a rotary evaporator at 35 °C. The dried sample was dissolved into 0.2 mL benzene. The solution was separated by a thin layer chromatography plate using benzene. The methyl ester of stearic acid was used as the marker probe. The zone, which consisted of methyl ester NSE, was extracted into benzene. Dried NSE powder was resuspended in ethanol to prepare 20 mM stock solutions. Serial dilutions of NSE were prepared in cell culture medium ex tempore before adding to cell culture.The human lung cancer cell line SW1573 and its ABCB1-overexpressing subline 2R160 were generously donated by H. Broxterman , human acute promyelocytic HL-60 leukemia cells and its ABCC1-overexpressing subline HL-60/adr were generously donated by M. Center . The human breast adenocarcinomas MCF-7 and MDA-MB-231, the human T-leukemia Jurkat, the murine leukemia L1210, the human cervix carcinoma HeLa and the pseudonormal human HEK293 kidney cells were purchased from the American Type Culture Collection . The murine NK/Ly lymphoma cells were obtained from the Kavetsky Institute of Experimental Oncology, Pathology, and Radiobiology, National Academy of Sciences of Ukraine .v/v) heat-inactivated fetal bovine serum  and 50 μg/mL gentamicin  in a humidified atmosphere containing 5% CO2 at 37 °C. All other cell lines were cultured in FCS- and gentamycin-supplemented RPMI-1640 medium. Cells were subcultured every 72 h at the rate of 5 × 105 cells per 1 mL of culture medium for suspension cultures and 5 × 104 cells per 1 mL of culture medium for substrate-dependent cultures. For morphologic assessment, cells were checked under an inverted microscope IB-100 .SW1573 and SW1573/2R160 cells were cultured in DMEM medium  supplemented with 10% (5 per well (suspension cell lines) and 105 per well (adherent cell lines). Short-term (24 h) cytotoxic effects were studied under an Evolution 300 Trino microscope  after cell staining with trypan blue dye (0.1%).For cytotoxicity studies, cells were seeded into 24-well tissue culture plates  at a concentration of 5 × 10For cell death analyses, cells were stained with APC-conjugated Annexin V and propidium iodide (PI) using an apoptosis detection kit , according to the manufacturer’s instructions. In particular, 24 h after the addition of various concentrations of NSE and Dx, Jurkat T-leukemia cells were centrifuged at 1200 rpm, washed twice with PBS, and incubated for 15 min in Annexin V binding buffer  containing 1/50 volume of APC-conjugated Annexin V solution and PI (50 µg/mL). Then, samples were diluted 2 times by appropriate volume of Annexin V binding buffer  and immediately measured using 670/14 nm (APC) and 575/26 nm (PI) filters of BD LSR Fortessa flow cytometer . Analysis of the obtained results was carried out using Cell Quest Pro software .6 cells were collected, pelleted by spinning at 1000 rpm, 4 °C for 5 min, resuspended in 1 mL of cold PBS, and fixed by adding drop-by-drop 4 mL of −20 °C absolute ethanol. On next day, fixed cells were centrifuged again, and cell pellet was resuspended in 1 mL PBS. Then, 100 µL of 200 µg/mL DNase-free, RNase A  was added to the cell suspension and incubated at 37 °C for 30 min. After this, 100 µL of 1 mg/mL propidium iodide (PI) was added to samples, which were incubated at room temperature for 5–10 min and then analyzed on a FACScan flow cytometer . Cell cycle analysis was carried out using Cytomation Summit Software v3.1 .Impact of drug treatment on the cell cycle distribution of Jurkat cells was assessed according to protocol described by Walker et al. . After d2O2)  and dihydroethidium   in Jurkat T-leukemia cells. To this end, the cells were either solvent-treated or incubated with various concentrations of the new compounds and then treated with 10 μM of either DCFDA or DHE at 37 °C for 30 min. After incubation with the dyes, cells were washed with PBS and immediately analyzed with the FL1 (DCFDA) or FL2 channel (DHE) on a FACScan flow cytometer .Cellular levels of reactive oxygen species (ROS) were measured by dyes dihydrodichlorofluorescein diacetate  at 37 °C. Spare dye was removed by PBS washing and cell-associated fluorescence was measured using 605/12 nm filter (for aggregate red form) and 530/30 filter (for green monomeric form) of BD LSR Fortessa flow cytometer . Analysis of the obtained results was carried out using Cell Quest Pro software .Breakdown of ΔΨm mitochondrial membrane potential was determined by FACS analysis using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) . Briefly, 105 cells/well) were seeded in 24-well tissue plates , and after 24 h incubation were treated with various doses of studied compounds. Intracellular Dx was detected using 575/26 nm filter in BD LSR Fortessa flow cytometer , and quantified using Cell Quest Pro software .Accumulation of free Dx, Dx-PC, and Dx-PC-NSE was measured by flow cytometry. Jurkat T-cells  for analysis of nuclear chromatin condensation. Subsequently, cells were washed in Hanks′ Balanced Salt solution , and cover glasses with living cells were placed on slides. Cytomorphological studies were performed using a Carl Zeiss AxioImager A1 fluorescent microscope . Image analysis was performed with Image-Pro 7.0 software .HeLa cells were seeded on cover glasses in 24-well plates  at a concentration of 106 cells, vortexed, and centrifuged. Protein concentrations in supernatants were measured, as described [L/S (sc-634) , caspase-9 (BD 556585) , β-actin , and monoclonal mouse antibodies against PARP-1 (BD 556434), FADD (BD 556402), Mcl-1 (BD 559027), caspase-2 (BD 611022), caspase-8 (BD 551242) , caspase-10 (M059-3)  for 12 h at 4 °C with slow shaking. Dilution for primary antibodies was 1:1000 in 5% BSA, 0.1% PBS-Tween, except for antibodies against β-actin (1:5000), as recommended by supplier. After incubation with primary antibody, the membrane was washed three times for 5 min in PBS with 0.1% Tween 20 and then incubated in 1:5000 dilution of secondary anti-rabbit IgG horseradish peroxidase-linked antibody  for 1 h at room temperature. The membrane was washed 3 times with PBS-Tween 20 for 5 min, and proteins that bound antibodies were visualized by incubation of the membrane for 1 min in ECL buffer , 2.72 mM cumaric acid  and 0.01% H2O2 in 0.1 M Tris-HCl (pH 8.5), and exhibition for 10–15 min to an X-ray film . Relative amounts of protein in the electrophoretic bands were quantified by using ImageJ Analysis software . Protein normalization was conducted for β-actin levels in the same samples.Western blot analysis was used to evaluate expression of proteins involved in cell death-signaling pathways in Jurkat cells after treatment with Dx, Dx-PC, or Dx-PC-NSE at various time points . The cells were washed twice on ice with PBS before addition of lysis buffer , 20 µL per 10escribed . Then, tad libitum. Mice were checked on a daily basis for health and well-being. The procedures of all in vivo studies gained ethical approval from the BioEthics Committee of the Institute of Cell Biology, NAS of Ukraine (protocol N9/2014 dated 1.09.2014). The study on the lipid profile was carried out with the approval of BioEthics and Biosafety Committee of the Palladin Institute of Biochemistry of NAS of Ukraine (Protocol #08/09-2015).The male Balb/c, DBA/2, and BDF1  mice used in this work were bred at the Institute of Cell Biology NAS of Ukraine and housed at the animal facility of the institute in a temperature-controlled environment with 12 h light/dark cycles, standard food ration for rodents  and water 5 tumor cells taken from a donor animal on days 6–7 of tumor development and washed with PBS.NK/Ly lymphoma cells were maintained by intraperitoneal transplantation of 0.2–0.3 mL of ascites fluid (0.5–1 million cells) on days 7–8 of lymphoma growth from a tumor-carrying animal to a recipient animal of the Balb/c strain. L1210 leukemia was passaged in hybrid BDF1 mice by injection of 1 × 106 cells) were injected i.p. per animal. The test compounds in different doses  were administered i.p. in a volume of 0.2 mL  per mouse every other day from the 2nd to the 20th day after tumor inoculation .Animal treatment studies were done in Balb/c mice and consisted of 5 separate experiments for NK/Ly lymphoma, due to large number of tested drug concentrations. For every dose of compound , animals were divided into four experimental groups with 6 animals in each. In a separate experiment, the anticancer activity of PC with or without immobilized NSE was studied in detail . To this end, 1 mL of NK/Ly lymphoma cells in HBSS  were inoculated i.p. to BDF1 mice, the course of treatment consisted of 8 injections of studied compounds in different doses , which were administered i.p. per mouse every day from 2nd to the 9th day after tumor inoculation according to standard protocol [Animal studies on L1210 leukemia were done in BDF1 mice in 4 separate experiments. For every dose of compound , animals were divided into four experimental groups with 6 animals in each. In a separate experiment, anticancer activity of the PC with or without immobilized NSE was studied in detail, and in this case, animals were divided into 10 experimental groups with 4 animals in each. L1210 leukemia cells (10protocol . The bod2-EDTA solution) as an anticoagulant. Hematological profiles in the inspected mice, including counts for hemoglobin level, red blood cells, platelets, white blood cells, relative neutrophils (%), lymphocytes (%), and monocytes (%) were studied using a 5-Part automated hematology analyzer DF51Vet  according to manufacturer’s instructions.For hematological analysis, healthy Balb/c mice  were treated with 5 injections of 4 mg/kg dose of Dx i.p. in a volume of 0.2 mL  per mouse every other day . Dosages of Dx-PC and Dx-PC-NSE were re-calculated to ensure the final dose of Dx injected into mice (4 mg/kg) was equivalent to free Dx. PC and PC-NSE were injected into animals in the same dose of the polymeric carrier, as in the system loaded with Dx. Blood samples (100 μL) were collected via retro-orbital bleeding in microtubes containing 5 µL of 10% ethylenediaminetetraacetic acid disodium salt solution (Nag for 10 min at 4 °C and stored in a freezer at −20 ℃ until use. Measurement of activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as concentrations of creatinine and urea, were conducted on a semi-automatic biochemistry analyzer BS-3000M  using diagnostic kits from Cormay S.A. . All measurements were performed according to the manufacturer’s instructions.For the serum biochemical analysis, blood samples were collected via retro-orbital bleeding into microtubes without anticoagulant, and serum was separated by centrifugation at 190× The animals were randomly divided into 5 groups, 6 animals per group. The mice of the control group received water, and the animals of the second group aqueous suspensions of the NSE at a dose of 50 mg/kg per os for 7 days. The third group of mice was treated with Dx i.p. at a dose of 2.5 mg/kg  in order to reach a cumulative dose of Dx of 7.5 mg/kg. The animals of the fourth group received NSE per os for 7 days at a dose of 50 mg/kg together with Dx i.p. at a dose of 2.5 mg/kg three times in an interval of one day. The fifth group of mice received NSE per os for 7 days at a dose of 50 mg/kg, after that, animals were treated with Dx i.p. at a dose of 2.5 mg/kg three times with an interval of one day. The animals of all groups were decapitated under ether anesthesia on the 12th day of the experiment. The mice’s hearts were used for analysis of the phospholipids profiles. Briefly, the content of heart phospholipids was estimated using thin-layer chromatography. The total lipids from the heart homogenates were extracted by Bligh and Dyer method . The lipt-test. Statistical significance was set at p ≤ 0.05. Evaluation of LC50 values of each compound was done by built-in functions of GraphPad Prism  vs. normalized response–variable slope). For analysis of animal survival, Kaplan–Meier curves were built in GraphPad Prism, and significance to other treatments was calculated by Mantel–Cox test. Statistical analysis of biochemical indices in mice was calculated using one-way ANOVA and Tukey’s multiple comparison test.All in vitro experiments were performed in triplicate and repeated three times. Statistical analysis of data was conducted in GraphPad Prism 8.0 software  using one-way ANOVA and Tukey’s multiple comparison test or Student’s 50 around 50 µM (50 = 30 μM) than in estrogen-sensitive MCF-7 cells (LC50 = 50 μM)  A. These 1 phase of the cell cycle, that was demonstrated by FACS analysis (1) cells were observed in these studies, despite a statistically significant decrease in Jurkat cell number under treatment with high doses of NSE , while such effects were observed upon treatment with low Dx doses  and high doses of NSE (20 µM) A. For co2O2 and O2− in Jurkat cells treated with LC50 dose of Dx (0.5 µM) in combination with NSE in different doses . However, no significant inhibitory effect of NSE on hydrogen peroxide or superoxide anion levels, considered as the main cause of Dx-induced cardiotoxicity, were found -5-methyl-1-hexen-3-yn-co-glycidyl methacrylate)-graft-PEG and used this formulation as a platform for targeted Dx delivery.50 values of Dx-PC-NSE conjugates were found to be 2-25-fold lower than the one of free Dx and 1.5-10-fold lower than those of the Dx-PC complex (50 = 0.85 µM) compared to Dx (LC50 = 11.02 µM), while immobilization of Dx on PC without NSE had no impact on its MDR-circumventing properties (LC50 = 9.32 µM). In order to verify these data, HL-60/adr (ABCC1+) cells were used delivering similar results with the Dx-PC-NSE complex  or by the enhanced entry of an anticancer drug into the cytoplasm or nuclei in a way not vulnerable to ABC transporter-mediated efflux from the membrane bilayer.In order to verify whether NSE is really capable of facilitating the entry of the Dx-polymeric carrier to treated cells, a cytomorphological study on HeLa cells was carried out. It was shown that after 6 h, Dx-associated fluorescence (2 μM) was detected in the cytosol of treated cells, but not in the nuclei . We obsew/v both of Dx and NSE, molar ratio Dx: NSE 1: 1.82), the drug fluorescence was detected in the nuclei of treated cells even at the lower dose of 2 μM. Thus, the presence of NSE in the composition of the Dx-loaded PC significantly (2-fold) enhanced the penetration of this drug to the nucleus more efficiently than the carrier itself. This is well reflected by the decreased LC50 values of Dx-PC-NSE in comparison to Dx and Dx-PC , was prepared. Subsequent biological experiments showed that the activity of this Dx-PC-NPE complex (16 methylene groups in lipid chain) towards MCF-7 cells was 20–30% lower than that of Dx-PC-NSE (18 methylene groups in lipid chain)  and thus increased apoptosis induction. In order to confirm this hypothesis, another d chain) . This efIn order to reveal if this phenomenon is somehow connected to enhanced entry of NSE-functionalized polymeric carrier with Dx into the cells or to enhanced Dx release by PC-NSE, we have performed quantitative analysis of Dx release from PC and PC-NSE in cell-free systems and FACS studies of Dx entry into Jurkat T-cells in time-dependent mode. As one can see in 2 peak, Dx-PC-NSE in the same concentration caused a complete G2-block in Jurkat cells. The same effect was achieved when Jurkat cells were treated with a five-fold higher dose of Dx (0.5 µM). However, when the Dx-PC-NSE complex was used at equimolar concentrations, it led to further changes in cell cycle distribution, primarily an increase in the S-phase fraction , which was observed at both low (2 µM) and high Dx doses (4 µM) in Jurkat cells . However, in the Dx-PC group, only 16% of animals were alive 90 days after treatment, while in the Dx-PC-NSE group, all animals exhibited no signs of the tumor until the end of the experiment, thus indicating a cure from this tumor. The same effect with Dx used alone was observed only when a cumulative dose of 10 mg/kg was applied A. Thus, In order to confirm the results obtained with NK/Ly lymphoma, another murine tumor model—L1210 leukemia—was used. In these experiments, a dose of 0.2 mg/kg Dx was insufficient to achieve a therapeutic effect, although even in this case, treatment of the animals with Dx-PC-NSE led to an improvement of their lifespan from 18 to 22 days compared to the effect of free Dx. When a 0.5 mg/kg dose of Dx was used, the lifespan of animals increased proportionally from 10.5 to 23 days, with maximum overall survival of 24 days. Dx-PC-NSE increased the life span to 33 days and 33% of animals showed signs of complete clinical response (life expectancy > 90 days) B. In theSummarizing, co-immobilization of Dx and NSE on PC leads to an approximately two-fold increase in the therapeutic activity of Dx in two different experimental murine tumor models, leading even to complete remissions in several animals. The main reason for the observed effect is the co-immobilization of the NSE and Dx on PC since the therapeutic activity of Dx-PC only slightly differs from such activity of the Dx used alone.In order to reveal if binding of Dx to NSE-polymer complexes, besides enhancement of therapeutic activity, may also lead to a decrease in its main side effects , blood analysis of healthy Balb/c mice treated with sub-lethal dose of Dx (20 mg/kg) was carried out in a time-dependent manner. Dx led to a two-fold decrease in the white blood cell count already 15 days after treatment, that was accompanied by neutrophilia and lymphocytopenia . ImmobilNo statistically significant changes in the levels of creatinine and urea were observed in this experiment, indicating the absence of considerable kidney damage by the compounds at the concentrations used. However, AST activity was significantly elevated in the blood of Dx-treated mice, suggesting severe liver and heart damage. In contrast, Dx-PC-NSE did not induce these negative side effects of Dx .In another experiment, the phospholipid profile in the heart tissue of mice under treatment with Dx and/or NSE was investigated. A significant decrease in the content of cardiolipin (DPG) and sphingomyelin (SM) was detected, while the levels of the phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC) were considerably increased under by Dx treatment. No changes were detected in the content of the phosphatidylcholine (PCh) and phosphatidylethanolamine (PE) under treatment with Dx . It shouThus, we demonstrated that the application of Dx-PC-NSE at least partly prevents the negative side effects caused by the Dx in a short time interval, supporting our initial hypothesis about the dual role of NSE in mediating the anticancer potential of Dx. On the one hand, the lipophilicity of the NSE may speed up the entry of Dx inside tumor cells and nuclei even in MDR cells, leading to enhanced apoptosis induction. On the other hand, the tissue-protective activity of NSE would lead to better protection of the liver and hematopoietic system of mice at sub-lethal concentrations of Dx.®  or Myocet® (liposome-based non-PEGylated Dx) have shown significantly decreased side effects, compared to original Dx, but there was no distinct increase in its therapeutic efficiency [Up to now, Dx remains one of the most widely used anticancer drugs for the treatment of cancer of different types and tissue origins . Howeverficiency . That isIn previous studies, we revealed that the use of synthetic comb-like PEGylated polymers reduced the effective dose of Dx but had little effect on its adverse effects in vivo . Here, wEarlier, it was demonstrated, that NSE administration per os led to a partial normalization of the activities of superoxide dismutase and glutathione peroxidase in heart tissue of rats, which were severely decreased under Dx treatment . Similar50 in the 30–50 µM range depending on the cell line, while pseudonormal human kidney embryonal HEK293 cells were found to be more resistant to NSE’s action. This indicates the selectivity of NSE towards malignant cells in vitro. Inhibition of tumor cell growth by NSE was accompanied by an increase in cell size as well as cell cycle arrest in the G1 phase. Despite dose- and time-dependent decrease in cell number under NSE treatment, it led to almost complete inhibition of basal level of apoptosis in the population of Jurkat cells at 48 h. Thus, we suggest that the main mode of cytostatic action of the NSE in vitro might be based on the enhanced incorporation of this lipid-like compound into the plasma membrane of target cells that may lead to inhibition of their proliferation. NSE also demonstrated a weak antioxidant activity at 24 h treatment of Jurkat T-leukemia cells, but this effect was not statistically significant.To select proper concentrations of NSE for PC immobilization, we performed in vitro studies of its cytotoxic activity towards several tumor cell lines as compared to pseudonormal cells. NSE possesses weak cytotoxic activity towards tumor cells with an average LC50 dose (0.5 µM) of Dx. Thus, we assume that the antioxidant activity of the NSE per se is not sufficient to modulate its action towards Dx-induced redox cycling. That is why further efforts were made to increase the bio-availability of the NSE via its immobilization to PC together with the Dx. Several formulations with a variable ratio of Dx and NSE were synthesized, and the most stable composite with 1 part of Dx and 1 part of NSE w/w  was selected for further studies.Co-treatment of human Jurkat T-leukemia cells with various doses of Dx and NSE has revealed the weak antagonistic effect of these compounds only at low doses  for 48 h, revealed by trypan blue exclusion assay and Annexin V/PI double staining. We did not observe any impact of the NSE on the levels of hydrogen peroxide and superoxide radicals in Jurkat T-cells when applying an LCp < 0.05) of superoxide production in Jurkat T-leukemia cells at early time points after treatment with various doses of Dx-PC-NSE, but not with Dx and Dx-PC. This may suggest partial inhibition of Dx-induced mitochondrial depolarization by NSE at early time points that might be a reason for its antioxidant activity in vivo, observed by us earlier [We detected that co-immobilization of Dx and NSE significantly increased the potential lipophilicity of Dx-PC-NSE, resulting in its enhanced uptake in vitro. Moreover, drug release studies had revealed an increased release rate of Dx from PC-NSE, starting at 75 min timepoint, which resulted in a further boost of Dx entry inside Jurkat cells at 90 min, as revealed by FACS analysis of Dx uptake. This, in turn, led to an elevated proapoptotic and cytotoxic activity of Dx towards a series of tumor cell lines, and also is suggested the main reason for partial circumvention of ABCC1 and ABCB1-driven resistance of cancer cells in vitro by Dx-PC-NSE, but not Dx-PC. On the other side, immobilization of the NSE to PC also led to enhanced antioxidant properties. The increase in the anticancer activity of Dx-PC-NSE complexes was accompanied by a significant decrease  led to 100% recovery of all mice in the experimental group, thus, confirming our data, obtained from the NK/Ly lymphoma model.In order to reveal potential mechanisms of an enhancement of Dx therapeutic action by the NSE immobilized on the polymeric carrier, we conducted in-depth functional studies of the hematopoietic system, as well as liver, heart, and kidneys of healthy mice treated with sub-lethal doses of free Dx (20 mg/kg) and immobilized to PC with or without the conjugated NSE. Dx treatment caused severe leucopenia, thrombocytosis, lymphopenia, and neutrophilia, while the application of Dx-PC-NSE was capable of partial reduction of these adverse effects. Surprisingly, Dx-PC led to an even stronger increase in monocyte levels than free Dx, which may explain a decreased survival of tumor-bearing animals treated with Dx-PC due to the development of thrombosis . Recent We also observed a two-fold elevation of AST activity (a marker of hepatotoxicity) in blood serum by treatment with a 20 mg/kg dose of Dx, while the application of Dx-PC-NSE did not induce liver toxicity. The same effect, but to a lower extent was observed for ALT, which is a known biomarker of the functional status of the liver. The absence of statistically significant changes in creatinine and urea levels in the blood of mice under these treatments is in agreement with previous data on the tissue-protective activity of the NSE ,41,43,44It is known that the cardiotoxic effect of Dx is related to its high affinity to DPG, a unique phospholipid located in the inner membrane of mitochondria . This liSummarizing, co-immobilization of the NSE and Dx on a micellar polymeric carrier leads to a distinct increase in Dx uptake into tumor cells with an enhancement of the anticancer activity both in vitro and in vivo. In parallel, an increase in the antioxidant activity of the NSE takes place, when it is applied in a composite with this drug delivery platform. Such composite is capable of effective protection of heart and liver tissues of mice with transplanted NK/Ly lymphoma and L1210 leukemia from adverse effects of Dx, finally leading to the final cure of treated animals. Altogether, the obtained data suggest a high potential of Dx-NSE nanoconjugates in clinical oncological practice.A unique bi-functionality of the synthetic cannabimimetic NSE was revealed. It demonstrated a proapoptotic activity on tumor cells in vitro and suppressed tumor growth in vivo. At the same time, in complex with the polymeric carrier, NSE showed a bioprotective effect against the general toxicity of Dx and particularly its cardiotoxicity. Potential mechanisms responsible for such a bioprotective role of NSE were elucidated.


Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.We investigated the bacterial food digestion and accumulation in wild-type adult  Host–microbiota interactions have a profound and wide-ranging impact on the health, aging and diseases in humans ,2,3,4,5.C. elegans nematode is a genetically tractable model organism for studying host–microbiota interactions and, in particular, bacterial pathogenesis, innate immunity and aging [E. coli) [E. coli is dynamic and changes over time. In the larval stages, the bacteria are thought to be simply a food source, which are killed by the pharyngeal grinder and, thus, no live bacteria in principle reach the intestine. In young adult worms, few intact bacteria manage to pass through the pharyngeal grinder without being damaged and form a community in some areas in the intestine and thus establish a commensal relationship with the worm [The nd aging . As the nd aging ,12,13. Tnd aging ,9. In laE. coli) . The relthe worm . 50 , the average number of colony-forming bacteria per worm at a given time point, and the gene expression of the worm that is measured either through fluorescence microscopy, blotting techniques or quantitative real-time polymerase chain reaction (qRT-PCR) analysis. We can use TD50 as a simple way to compare the pathogenicity of different bacteria assuming that the considered bacteria support the worm adequately from a nutritional standpoint. For example, an adult wild-type worm population feeding on pathogenic P. aeruginosa PAO1 has TD50 = 2.84 days [E. coli OP50 has TD50 = 12.93 days [P. aeruginosa PAO1 shows that this bacterium exhibits a higher degree of pathogenicity compared to E. coli OP50. Despite the extensive research on non-pathogenic E. coli OP50 [P. aeruginosa [P. aeruginosa, little is known about the impact of the bacterial food source on the digestion/absorption of bacterial material by the worm through its intestine. The interaction between the bacteria and the worm is often characterized in terms of TD.84 days , while t.93 days . Both of.93 days ,16,17. Holi OP50 ,17,18,19ruginosa ,26,27,28C. elegans was reported for the first time in 2007 [The use of microfluidics for studying  in 2007  and, sin in 2007 ,32,33,34 in 2007 . General in 2007 ,35 or lo in 2007 ,40,41,42C. elegans infection assay was proposed by Yang et al. [Staphylococcus aureus and, afterward, the efficacies of the antibiotic compounds as a function of their doses were assessed through survival curves. As a result, this device is mostly suitable for in vivo antibiotic compound screening. Lee et al. [C. elegans killing assays. The worm and the pathogen of interest could be loaded in a large chamber, where the motility and survival of the worms and the expression of irg-1::GFP could be monitored in vivo through low-resolution imaging. However, due to the lack of compartments, the identities of the worms could not be tracked over time. The first microfluidic device for implementing a g et al.  in 2013.e et al.  reportedC. elegans. The device featured four separate lanes each of which comprised five compartments. A total of 1 to 3 worms were loaded into each compartment where they were fed fluorescent-tagged bacteria. Through low-resolution fluorescence imaging and subsequent image analysis, the spatiotemporal evolution of intestinal bacterial load of the freely moving worms could be obtained. In this way, the periodic behavior associated with ingestion of bacteria and its clearance from the intestine was monitored and its temporal periodicity was determined. The worms could also be immobilized in the tapered structures present in the compartment, which rendered them incapable of feeding. Afterward, low-resolution fluorescence images were acquired from these worms to obtain the time constant of the bacterial transition. In a subsequent work, Viri et al. [Viri et al.  reportedi et al.  improvedi et al.  such thai et al. , they usC. elegans worms with an unprecedented resolution. To achieve this, we relied on loading the worm population of interest on a simple microfluidic chip where the worms could be subsequently fixed for high-resolution imaging. We preferred this approach to the more conventional method whereby previously anesthetized worms are mounted on an agarose pad before they are observed [SDCMs offer superior resolution compared to widefield microscopes by incorporating pinholes that block the light signal from out-of-focus planes. Therefore, to improve upon the previous works by Viri et al. ,46, we aobserved ,48. Whil®), 4-inch 550 µm Si wafers, 5-inch Chromium/soda–lime glass masks and trimethylchlorosilane (TMCS)  were acquired from the Center of MicroNanoTechnology (CMi) at EPFL . Kayaku Advanced Materials (KAM) SU-8 3050 was bought from Micro Resist Technology GmbH . Glass coverslips of 45 mm × 70 mm × 170 µm were obtained from Biosystems Switzerland AG . Saint-Gobain Tygon™ ND 100–80 Tubing (inner diameter and thickness of 0.02 inch and outer diameter of 0.06 inch) was purchased from Fisher Scientific . NGM plates were ordered from the Solution Preparation Facility at EPFL. In order to prepare S-basal, 5.85 g of NaCl, 6 g of KH2PO4 and 1 g of K2HPO4 were first dissolved in H2O until a final volume of 1 L was realized. This solution was subsequently sterilized via autoclaving and, afterward, 1 mL of cholesterol solution (5 mg mL−1 in ethanol) was added using a sterile technique to obtain S-basal. In order to make S-medium, 500 µL of 1 M potassium citrate (pH 6), 500 µL of trace metals solution, 150 µL of 1 M CaCl2 and 150 µL of 1 M MgSO4 were added using a sterile technique to 50 mL of S-basal. The materials required for the preparation of S-basal, S-medium and lysogeny broth (LB) for bacteria culture and tetracycline were purchased from Sigma-Aldrich Chemie GmbH . The Laboratoire des Polymères (LP) at EPFL  kindly provided 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS), which was used for the fixation of C. elegans.Polydimethylsiloxane (PDMS) Sylgard 184 (DowC. elegans wild-type (WT) Bristol N2 strain in our experiments. The worm populations were synchronized by suspending gravid adult worms (that can have slightly different ages) overnight inside a 50 mL falcon tube containing 8 mL of S-medium. During this time, due to the lack of food, the L1s that hatch from the eggs laid by the adults underwent developmental arrest [E. coli OP50 and maintained there for 46 h at 22 °C until they became adults and were thus ready for the experiments. We used l arrest  and, thuE. coli OP50 was cultured overnight in LB at 37 °C on a shaker. Red Fluorescent Protein (RFP)-labeled E. coli OP50 was kindly provided by the C. elegans Ageing Laboratory at the University College London. Plasmid pRZT3::dsRED, which also contained genes for tetracycline resistance, was used to transform E. coli OP50 and produce the RFP-labeled E. coli OP50. RFP-labeled E. coli OP50 was cultured in LB containing 10 µg/mL of tetracycline overnight at 37 °C on a shaker. RFP-labeled P. aeruginosa PAO1, which was kindly provided by the Laboratoire des Polymères (LP), constitutively express mCherry (attTn7::miniTn7T2.1-Gm-GW::PA1/04/03::mCherry) in the bacterial cytoplasm. RFP-labeled P. aeruginosa PAO1 was cultured overnight in LB at 37 °C on a shaker.The microfluidic chip was designed in Clewin 4.0  and through standard mask fabrication techniques, and the designed layout was then transferred to a chromium/soda-lime glass mask. Next, the mask was used to create a 75 µm high SU-8 mold on a Si wafer using standard soft photolithography techniques. To facilitate the separation of the polydimethylsiloxane (PDMS) chip from the SU-8 mold, the mold was treated with trimethylchlorosilane (TMCS) and, afterward, PDMS with a base-to-curing agent ratio of 10:1 was cast onto the mold and cured for 2 h at 80 °C inside an oven. Once the PDMS cured and cooled down, it was cut according to the design of the device and the resulting PDMS block was removed from the mold. Afterward, using a biopsy punch, inlets and outlets were made in the PDMS block. The PDMS block and a glass coverslip measuring 45 mm × 70 mm × 170 µm were bonded together, utilizing oxygen plasma surface activation, to create the microfluidic device. The microfluidic chip was then placed on a hotplate for 10 min at 80 °C to improve the bonding strength.Raw z-stack fluorescence images were processed in IMARIS (version 9.9.1). The software constructs three-dimensional (3D) images from z-stack fluorescence images, which can be further analyzed using the built-in toolkits. The results of image analysis by IMARIS are exported to excel files that are processed in an automated fashion in MATLAB software (version 2022a). The processing involves selecting the data of interest and performing basic arithmetic operations on them. The processed data can then be easily imported in GraphPad Prism software (version 9.5.0) to be plotted and tested for statistical significance. The one-tailed Mann–Whitney test was utilized to establish statistical significance.We utilized two different microscopes in our experiments. A Zeiss Axio Imager.M2 operated in brightfield mode and equipped with a 2.5× Zeiss objective was used for preparing the microfluidic chip, loading worms on the chip and their subsequent fixing. High-resolution z-stack fluorescence imaging was realized using Visitron’s CSU-W1—a spinning disk confocal microscope equipped with (i) two laser light sources for fluorescence imaging in the RFP and the Green Fluorescent Protein (GFP) channels, running at wavelengths 561 nm and 488 nm, respectively; (ii) an Olympus U PLAN S APO 60×/1.42 NA oil immersion objective; (iii) emission filter cubes ET605/70m and ET525/50m, by Chroma Technology , for the RFP and the GFP channels, respectively; and (iv) a Hamamatsu ImagEMX2 electron-multiplying CCD (EMCCD) camera. The imaging was carried out with the following parameters: exposure time of 60 ms, laser power for both the RFP and the GFP channels of 50%, gain of the EMCCD camera set to 200, pinhole size of 50 µm and z-stack imaging in a 50 µm range with a step size of 0.2 µm.Our microfluidic chip consists of five identical lanes, the layout of which can be seen in The lanes are 2 cm long, 1300 µm wide and 75 µm high, the last of which makes the device capable of accommodating adult worms. Furthermore, each lane features two filter structures whose functions are to confine the loaded worms to the right side of the channel and provide control over the number of worms present on the chip. An example of a fabricated microfluidic device is shown in −1. Once around 10–15 worms are present in the channel, an Eppendorf tube containing a 4% PFA fixation solution is placed at the outlet and, by aspirating with a flow rate of 80 nL s−1, the fixation solution is introduced in the channel. The worms become fixed by staying in contact with the fixation solution for about 5 to 10 min, after which the inlet and outlet tubings are cut and fastened and the microfluidic chip can be taken to an SDCM for high-resolution z-stack fluorescence imaging. The microfluidic chip is operated while being observed in the brightfield mode using a Zeiss Axio Imager.M2 equipped with a 2.5× Zeiss objective. Before worms can be loaded on the chip to be fixed and imaged, a chip initialization step should take place. Therefore, S-medium is first injected from the inlet to the channels using a neMESYS syringe pump . However, as the air in the channels is being replaced by S-medium, some bubbles of air are bound to form. To eliminate these, the outlet tubing is clipped and the channel is pressurized by injecting 10 µL of S-medium through the inlet. The increased pressure promotes the outward diffusion of air through PDMS and thus the channel is cleared from air bubbles within 15–20 min. Afterward, the outlet tubing is unclipped and is placed inside an Eppendorf tube containing the adult worms of interest suspended in S-medium. The worms can then be loaded on the chip d by usinC. elegans worms that have fed on either RFP-labeled E. coli OP50 or P. aeruginosa PAO1 during the first 4 days of adulthood via high-resolution z-stack fluorescence imaging . This procedure is repeated 10 times  and thus the number of E. coli OP50 present in S-medium is significantly reduced  requires fixation, the same worms cannot be studied at different time points. Additionally, while the adult worms are feeding (on either of the fluorescent bacteria), they also lay eggs that hatch into L1s. We wish to study the bacterial food digestion and accumulation in wild-type adult  imaging . First,  imaging a by suspdulthood b. At thi reduced c. The add 4 days d. As hig10. Afterward, the adult worms are transferred to a fresh NGM plate . It follows naturally that the steps described in P. aeruginosa PAO1, it should be mentioned that the lab  that provided us this bacterium used a 4% PFA solution as per their fixation protocol and hence, in good faith, we follow the same protocol.These L1s not only mix with the adult population, which later can become a source of confusion, but they can also rapidly deplete the NGM plate of bacterial food. Therefore, every 24 h, the adult worms are separated from L1s through serial dilutions e, which GM plate d seeded We start the imaging procedure with a low-magnification brightfield image a to firsOnce the hindguts of the worms of interest are identified, we carry out high-resolution imaging of the hindguts in the brightfield mode and the RFP and the GFP channels, an example of which is shown in The adult wild-type worms exhibit autofluorescence, which at least partly originates from the intracellular-lysosome-derived granules within the intestinal cells ,53.This autofluorescence signal can be observed in the Blue Fluorescent Protein (BFP), GFP and RFP channel . The exaE. coli OP50 or P. aeruginosa PAO1 reveal two distinct patterns for the bacteria-related fluorescence signal   b. This oous work , we expoed worms a, we alsed worms c. To ensE. coli-OP50-fed worms, however, is not applicable to the fluorescent P. aeruginosa-PAO1-fed worms, as the intensity of RDF is too high to be ignored. Additionally, as mentioned earlier, the fluorescence signal originating from the intact fluorescent P. aeruginosa PAO1 bacteria can be masked by the signal from the disrupted fluorescent P. aeruginosa PAO1 bacteria and, thus, a bacterial load analysis based on separating these two types of fluorescence signal will not be entirely meaningful. Consequently, we decide to consider the RDF in its entirety, including the fluorescent spots that are spread within it, as one object for analysis , which ranges from 2770 to 2900 across all the experiments. The threshold value used for defining the surface is set 600 higher than the background noise and thus ranges from 3370 to 3500.The approach used for fluorescent analysis b. In IMAThis surface can be then used to define RDF as the volume within which the fluorescence intensity is higher than the threshold value d. Once tE. coli OP50, IMARIS provides datasheets containing the volumes and the average intensities of every fluorescent spot in each worm. These datasheets are then imported into MATLAB software to be processed using basic arithmetic operations. For each worm, we determine the total volumes of the fluorescent spots. Additionally, we apply a background correction to the average intensities of the fluorescent spots by subtracting from them the background noise intensity. Afterward, a volume-weighted average of the background-corrected average intensities of all the fluorescent spots in each worm (referred to as “AVG Intensity”) is calculated. Thus, the bacterial load in every worm is proportional to the multiplication of the total volume and the “AVG Intensity” of the fluorescent spots. In the case of worms that have fed on RFP-labeled P. aeruginosa PAO1, IMARIS provides datasheets containing the volumes and the average intensities of the RDF in each worm that are similarly imported and processed using MATLAB. However, it must be remembered that in the case of RFP-labeled E. coli-OP50-fed worms, the bacterial load describes the total number of intact bacteria, while in the case of RFP-labeled P. aeruginosa-PAO1-fed worms, the bacterial load describes the Sum of Intact and Disrupted Bacteria (SIDB). Regardless, in both cases, the processed data are plotted in GraphPad, yielding The image analysis by IMARIS allows us to analyze the intestinal bacterial content with single-worm resolution. In the case of worms that have fed on RFP-labeled E. coli OP50. As there is a large variation in the data points, the use of log plot is necessary. As a result, no data points can be shown for worms whose intestines are free of bacteria (since log(0) is undefined). Instead, the number of worms devoid of bacteria on each day is mentioned inside the plot alongside the data points of the respective day. The absence of a number indicates the absence of worms devoid of bacteria on the respective day. This convention has been applied to plots in E. coli OP50 are devoid of fluorescent spots and thus intact bacteria in the first two days. Nonetheless, there are three worms on the second day in which the total volume of the fluorescent spots is significantly large. This shows the importance of single-worm resolution analysis since considering the average of the total volume of the fluorescents spots alone would imply the presence of a significant volume of fluorescent spots in all the worms. On the third day, there is a sharp increase in the number of worms whose intestines contain significant volumes of fluorescent spots and, on day 4, the intestines of all the worms contain significant volumes of fluorescents spots. Similar observations can be made when the “AVG Intensity” of fluorescent spots is considered in P. aeruginosa PAO1. It can be seen that on day 1, in about half of the worms, the total volume of the RDF is significantly large, and only in 25% of the worms is the total volume of the RDF very small . On day 2, there is an increase in the total volume of RDF and through comparison of intrapopulation variations among day 2, 3 and 4, it can be inferred that the volume of the RDF has already reached a saturation limit. This means that the intestinal bacterial load increases through the spread of intact bacteria in the intestine, rather than the formation of very dense bacterial clusters. Overall, it can be understood that until day 2, most of the worms are devoid of any intact bacteria and it is on day 3 and 4 where a significant number of intact bacteria are found in the intestine of every worm. However, considering the upper and lower limits of bacterial load variation among the worms on day 3 and day 4, it seems that the number of intact bacteria is approaching a saturation limit. E. coli-OP50-fed worms and RFP-labeled P. aeruginosa-PAO1-fed worms. Moreover, in the former case, the bacterial load is proportional to the number of intact bacteria, while in the latter case, it is proportional to SIDB. Additionally, we have not measured the fluorescence signal intensity per unit of either (intact) bacteria. As a result, direct quantitative comparison between the two groups should be avoided. Nevertheless, it is reasonable to assume that when the intestine of the worm is filled with either of the two bacteria, as indicated by reaching a saturation limit in E. coli OP50 and P. aeruginosa PAO1 bacteria present in the intestine are similar. This assumption allows us to compare the rate of bacterial colonization in the intestine of worms. In the case of RFP-labeled P. aeruginosa-fed worms, already on day 1, half of the worms contain a bacterial load similar to those of worms on day 4. However, in the case of RFP-labeled E. coli-OP50-fed worms, this takes place sometime between day 2 and day 3. The faster spread of RFP-labeled P. aeruginosa is also evident by the fact that after 2 days, the bacterial load reaches saturation, while for RFP-labeled E. coli OP50, the saturation can be thought to occur at the earliest on day 4. Therefore, in both cases, the intestinal bacterial load generally increases with the age of the worm, albeit more rapidly for worms fed on RFP-labeled P. aeruginosa PAO1. We must stress that we have used two different methods for the quantification of bacterial load in RFP-labeled C. elegans is a versatile model organism for studying bacterial pathogenesis and evolutionary conserved innate immunity pathways [E. coli OP50, a non-pathogenic bacterium, is used as a food source for the general maintenance of C. elegans worms [E. coli OP50 colonizes the intestine of the worm and thus becomes mildly pathogenic [P. aeruginosa is a known pathogenic bacterium that is capable of killing the worm through different means, and in particular, intestinal colonization [P. aeruginosa PAO1 is about 20% of those fed on E. coli OP50, which reflects the stark difference in the degree of pathogenicity [pathways ,18. E. cns worms . Howeverthogenic . On the nization ,26,27,28genicity ,16. P. aeruginosa infection in C. elegans via intestinal colonization, there are no systematic studies on the feeding behavior and the digestion in infected worms. We do know, however, that within 8 h of feeding on P. aeruginosa PA14—a more virulent strain compared to PAO1 we used in our study—the intestine becomes distended, containing few intact bacteria that are the source of putative Outer Membrane Vesicles (OMVs) that are also present in the intestine [P. aeruginosa PAO1 [E. coli showed no such signs [Despite the extensive research on ntestine . Additiontestine . Unfortuntestine . After 2ntestine . At 48 hntestine . A similosa PAO1  but wormch signs . P. aeruginosa-fed worms are related to the accumulation of disrupted bacteria we observed in P. aeruginosa PAO1 have shorter body lengths compared to those fed on E. coli OP50 [P. aeruginosa PAO1 reduces the rate of bacterial material absorption and/or defecation in the worms, which then results in the accumulation of nutrients/waste originating from disrupted bacteria in the gut. This hypothesis can be evaluated in future works, by observing how the time gap between the last feeding event and the fixation of the worms affects the pattern of the bacteria-derived fluorescence signals. In the present study, the time gap was 1 h, which was sufficient for E. coli-OP50-fed worms to fully digest the bacteria but not enough for P. aeruginosa-fed worms. Imaging the worms that are fixed with different time gaps  can eventually reveal the dependency of the digestion dynamics on the bacteria used as the food source. Whether and how the symptoms seen in oli OP50 , indicatE. coli OP50 or Salmonella typhimurium (S. typhimurium) SL1344 show that the number of colony-forming bacteria in the intestine generally increases with the age of the worm until it reaches a saturation limit, albeit earlier in the case of the more pathogenic S. typhimurium SL1344 [E. coli OP50 seem to approach a saturation limit on day 4, while for those fed on RFP-labeled P. aeruginosa PAO1, the saturation limit is reached on day 2. This observation can be at least partially explained, as we alluded to earlier, by the reduced metabolism in the worms fed on P. aeruginosa PAO1.Previous studies on worms fed on either m SL1344 ,18. We oEven though we used a simple microfluidic device in this work, the methods described in this work can be readily implemented in other types of microfluidic devices. Our group has already developed automated image and video processing techniques that can be used for in vivo extraction of phenotypes such as area, length, egg release rate and motility from the worms that are studied via microfluidic devices . As discC. elegans worms. We discovered that bacteria-derived fluorescence signals in the intestines of the worms fed on RFP-labeled P. aeruginosa PAO1 uniquely included a diffuse fluorescent background in addition to individual fluorescent spots that are also present in worms that were fed on RFP-labeled E. coli OP50. This observation indicates an intestinal accumulation of disrupted bacteria exclusive to RFP-labeled P. aeruginosa-PAO1-fed worms, which, when understood in the context of previous studies, hints at a reduced metabolism in such worms compared to those fed on RFP-labeled E. coli OP50. Furthermore, our single-worm resolution analysis showed that with both diets, the bacterial load in the intestine generally increases with age until it reaches a saturation limit. However, in the case of RFP-labeled P. aeruginosa-PAO1-fed worms, this limit was reached in 2 days compared to 4 days for RFP-labeled E. coli OP50, which further suggests a reduced metabolism in the former worms. The methods described here can readily be used to complement the scopes of existing microfluidic technologies.We have demonstrated the potential of on-chip high-resolution z-stack fluorescence imaging and advanced image analysis for elucidating the bacterial food digestion and accumulation in


Higher CD19 (B cell) counts favored vaccination response (adjusted odds ratio (aOR) 3.3 per 100 B-cells/microliters, p < 0.001), while ongoing mycophenolate mofetil (MMF) immunosuppression hindered it . By multivariable analysis, the time from transplant to first vaccine did not remain a significant risk factor. A total of 92% of non-responders received a third mRNA dose, achieving additional 77% seroconversion. Non-converters mostly received a fourth dose, with an additional 50% success. Overall, a cumulative seroconversion rate of 93% was achieved after up to four doses. (4) Conclusion: mRNA vaccines are promising for HSCT recipients as early as 3 months post-HSCT. A majority seroconverted after four doses. MMF usage and low B cell counts are risk factors for non-response.(1) Background: mRNA COVID-19 vaccines are effective but show varied efficacy in immunocompromised patients, including allogeneic hematopoietic stem cell transplant (HSCT) recipients. (2) Methods: A retrospective study on 167 HSCT recipients assessed humoral response to two mRNA vaccine doses, using the manufacturer cut-off of ≥7.1 BAU/mL, and examined factors affecting non-response. (3) Results: Twenty-two percent of HSCT recipients failed humoral response. Non-responders received the first vaccine a median of 10.2 (2.5–88.9) months post-HSCT versus 35.3 (3.0–215.0) months for responders ( The onset of the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has undeniably put enormous stress on worldwide health systems. Individuals with compromised immunity, including patients with hemato-oncological diseases who have undergone allogeneic hematopoietic stem cell transplantation (HSCT), have been shown to be vulnerable to this virulent pathogen. Research has highlighted that the mortality rate for allogeneic HSCT patients hovers around 16%, as reported in a recent study [The latter part of 2020 saw the advent of SARS-CoV-2 messenger ribonucleic acid (mRNA) vaccines, among which were the BNT162b2 and mRNA-1273 vaccines. The BNT162b2 and mRNA-1273 vaccines work by injecting a tiny fragment of the virus’s genetic code into the body. This triggers the production of the spike protein, which in turn activates the immune system. Once the immune system identifies the spike protein as an invader, it generates antibodies to combat it, thereby offering immunity against future infections with the virus ,5,6, whiFurther analysis showed that allogeneic HSCT recipients developed anti-spike (anti-S) antibodies after receiving two or three doses of the vaccine . This fiTherefore, in the current study, our primary objective was to examine and assess the risk factors that could potentially contribute to a lack of humoral response in allogeneic HSCT patients post-vaccination. Specifically, we aimed to scrutinize these factors following the administration of two BNT162b2 mRNA or mRNA-1273 vaccine boosters within a cohort of 167 allogeneic HSCT recipients. The findings of this research could hold significant implications for the overall public health strategy concerning vaccination protocols in immunocompromised patients, particularly those who have undergone allogeneic HSCT.This single-center retrospective research aimed to evaluate the rate of antibody response following a two-dose regimen of an mRNA vaccine for SARS-CoV-2. The study also sought to pinpoint the clinical and laboratory factors that might indicate a lack of effective antibody response in individuals who have undergone an allogeneic hematopoietic stem cell transplant (HSCT). The assessment of an effective antibody response was determined using a cut-off value set by the manufacturer at 7.1 binding antibody units (BAU)/mL or greater, as measured by the SARS-CoV-2 IgG II Quant assay from Abbott, Ireland ,11.The study population comprised all patients who had received an allogeneic blood stem cell transplant at the Ordensklinikum der Elisabethinen in Linz between 2 May 2003 and 12 March 2021 (163 of the 167 patients transplanted post-2011). The included participants were those who had undergone immunization (both first and second doses) by 12 October 2021 and had a minimum of 14 days between their second vaccination and titer measurement. In this study, the population was immunized using two types of COVID-19 vaccines: BNT162b2, which accounted for 88.0% (147 recipients), and mRNA-1273, which accounted for 12.0% (20 recipients). Several considerations were accounted for in the study. The last measured immunoglobulin (Ig) level and the most recently collected lymphocyte subtyping should not be older than 12 months for patients with a transplantation to first vaccination interval of up to 3 years. For patients with an interval of 3 to 5 years, the cut-off was increased to 18 months, and for those with an interval of >5 years the cut-off was 24 months. To account for potential confounding factors in recipients who recently received an immunoglobulin substitution, we ensured a minimum gap of 3 months since their last substitution.The study examined various factors, such as the time elapsed between HSCT and vaccination, the interval from vaccination to antibody level measurement, the ages of both the recipient and the donor, ongoing cancer treatments, specific immunosuppressive medications, the kind of graft-versus-host disease (GVHD) prevention used, gender, the relationship between the donor and the recipient, the type of vaccine administered, and certain immune system indicators. These immune indicators included counts of different types of blood cells ((cluster of differentiation) CD4, CD8, and CD19 and  NK cells) as well as levels of specific immunoglobulins .2 or even 103.Our immunocompromised patient cohort was among the first in Austria to initiate SARS-CoV-2 vaccination. Therefore, passive transfer via transfusion is very unlikely to have contributed to the measured titers in our cohort, particularly to titers > 10p < 0.2. Receiver operating characteristic (ROC) analysis was performed for CD19 cells with a cut-off estimation based on the Youden’s index. Comparative analysis of variables was conducted using the Fisher exact test and the Mann–Whitney U test. R statistical software was used for the analyses and plots used in this study [Statistical analyses included both univariate (response versus non-response) and multivariate logistic regression. Variables were excluded from the multivariate model until all variables were is study .The study involved 167 adult (>18 years) participants who had received an allogeneic HSCT. The results showed that 37 of these individuals, making up 22% of the total study group, did not show a measurable antibody response after completing a two-dose mRNA vaccine series for SARS-CoV-2.p < 0.001). Given that the immune system requires time to recover post-transplant, these findings were anticipated. The multivariate model revealed an adjusted odds ratio (aOR) of 1.02 .A detailed investigation into the vaccination non-responders revealed that the median time from HSCT to vaccination was 10.2 months, with a range of 2.5 to 88.9 months. In contrast, the median time interval for those who exhibited a response to vaccination (referred to henceforth as ‘responders’) was markedly longer at 35.3 months (range: 3.0 to 215.1 months). This difference in the time from HSCT to vaccination between non-responders and responders was statistically significant .Age, which can significantly influence a patient’s response to treatment, demonstrated a difference between the two groups. The median age of the total cohort was 58.10 years, ranging from 23.40 to 76.60 years. The age difference was statistically significant with a p-value of 0.06. When subjected to multivariate analyses, no significant disparities were evident . It should be noted that, in our study, a slightly higher number of males than females were evaluated. The study also observed differences in gender distribution, although these differences were not statistically significant with a p-value of <0.001. Similarly, CD8+ cells showed a median count of 488 cells/μL (33 to 4654) in responders and 295 cells/μL (44 to 2473) in non-responders, with a p-value of 0.01. For CD19+ cells, the median count was 295 cells/μL (0 to 1295) in responders, significantly higher than the 17 cells/μL (0 to 530) in non-responders, with a p-value of <0.001. NK cells showed a minimal difference, with median counts of 250 cells/μL (15 to 1979) in responders and 218 cells/μL (14 to 646) in non-responders, with a p-value of 0.05  in responders, compared to 154 cells/μL (ranging from 7 to 1007) in non-responders, with a highly significant  of 0.05 . These rp < 0.001). This observation underscores the pivotal role of B-cells in mediating a robust humoral response to vaccination. Upon conducting an ROC analysis, a threshold of 146.5 cells/μL yielded an area under the curve (AUC) of 89.3%  in responders and 436.00 mg/dL (161.00 to 2079.00) in non-responders (p < 0.001). Similarly, IgM levels were higher in responders, with a median level of 62.50 mg/dL (5.00 to 299.00), as opposed to 33.00 mg/dL (1.00 to 142.00) in non-responders, also with a highly significant p-value of <0.001. While these differences were statistically significant in the univariate analyses, they did not remain significant in the multivariate model. Additionally, by univariate analysis, we observed notable differences in immunoglobulin levels between responders and non-responders. For IgA, the median level was 92.00 mg/dL (ranging from 5.00 to 454.00) in responders, compared to 51.00 mg/dL (ranging from 1.00 to 1299.00) in non-responders, with a significant Out of the 37 non-responders, 34 (92%) received a third dose of one of the two available mRNA-based vaccines. This was carried out at a median of 189 days after the second vaccination (range: 56 to 273 days). One patient declined revaccination, while two other patients were not eligible for revaccination due to the progression of malignancy, which unfortunately proved to be fatal .Of the 34 individuals who did not respond to the initial two doses and went on to receive a third dose of the vaccine, 23 out of the 30 who were evaluated (77%) showed a successful antibody response, i.e., achieved seroconversion. For the seven patients who did not achieve seroconversion even after the third dose, six were administered a fourth mRNA vaccine dose. This was given at an average of 95 days after the third dose, and it resulted in a 50% additional seroconversion rate.Among the 23 patients who did achieve seroconversion following the third dose, 10 (44%) went on to receive a fourth mRNA vaccine. This was administered a median of 125 days after the third dose. The median antibody level for these 23 patients, as measured by the SARS-CoV-2 IgG titer, clearly increased from 290.8 BAU/mL after the third dose to 1750 BAU/mL after the fourth dose.By the end of the observation period, a total of 25 out of the initial 37 patients who failed the basic two-dose immunization were able to produce detectable levels of SARS-CoV-2 IgG antibodies. Thus, we demonstrated that the seroconversion rate of 78% after a two-fold immunization could be increased to a cumulative rate of 93% by two additional vaccines. This finding underscores the potential benefit of additional vaccine doses in inducing a detectable antibody response among HSCT recipients.p-value of 0.38. It is noteworthy that only 2 out of the 39 patients with confirmed COVID-19 infection required hospital admission. Specifically, one patient was from the non-responder group and the other was from the responder group. No deaths related to COVID-19 infections were observed in the study population. Admission to the intensive care unit was not needed for any of the patients.In our study cohort, we observed a total of 39 documented cases of COVID-19 infection. Among these, 11 cases were found in the non-responder group, while the remaining 28 cases were identified among patients with documented seroconversion. In the cohort of non-responders, the infection rate was observed to be 29.7%, whereas in the responder group, the rate was notably lower at 21.8%. Statistical analysis revealed that the difference in infection rates between the non-responder and responder groups was not significant, with a p < 0.001) in patients undergoing MMF therapy at the time of vaccine. On the other hand, calcineurin inhibitors (cyclosporine A or tacrolimus) as standalone immunosuppressive therapy did not show an impact on the outcome, with an aOR of 1.15 .The distribution of immunosuppressive regimens showed significant differences. While 60% of responders were off immunosuppression, a mere 18.9% of non-responders were off these drugs. Regimens like MMF combined with or without CNI were more prevalent in non-responders . Specifip = 0.15). However, there was a trend in the multivariable analysis when ruxolitinib was combined with calcineurin inhibitors, hinting towards a negative impact on vaccination outcomes with this combination, with an aOR of 0.12 .Further, it was observed that ruxolitinib when administered alone did not show a significant influence on the vaccination success, with an aOR of 0.25 .Inclusion of ATG for primary GVHD prophylaxis was a significant risk for non-response in the multivariate model, with an aOR of 0.26  than responders (30.8%). However, this difference was not statistically significant  received the BNT162b2 vaccine and 20 (12.0%) received the mRNA-1273 vaccine. In the responder group (n = 130), 115 (88.5%) received BNT162b2 and 15 (11.5%) received mRNA-1273. In the non-responder group (n = 37), 32 (86.5%) received BNT162b2 and 5 (13.5%) received mRNA-1273. The p < 0.001). This could not be supported by the multivariate model, where steroid therapy was not selected in the final model (A significant majority of responders (76.9%) were not on steroids, compared to only 35.1% of non-responders. This indicates that ongoing steroid treatment might negatively affect the response, a finding that was statistically supported  as a pivotal predictor for vaccine failure a year after transplantation . This fiIn light of the above, it is essential to discuss the key factors that might explain the differences between our findings and those presented by Schulz et al. . PrimariNo vaccine-related complications ,31 were Despite the valuable insights provided by this study, several limitations should be acknowledged. The retrospective design may have introduced bias, and the absence of a control group limits the ability to draw definitive conclusions. At the time of the described vaccination program, COVID-19 infections were still very rare in our highly isolated and protected vulnerable patient cohort. Since we also had documented individual infections in the cohort, only five patients had a documented infection before completing the described vaccination series. The wide range in the timing of vaccination relative to transplantation and titer measurement could have introduced confounding factors. The lack of long-term follow-up data restricts the assessment of the durability of the antibody response and the vaccine’s clinical effectiveness. While this study provides valuable insights into the humoral response to vaccination, it is important to note that it does not fully capture the complexity of the immune response to SARS-CoV-2, particularly the role of T-cell responses. Indeed, recent research underscores the robustness of T-cell responses to vaccination, even in the absence of detectable antibodies ,33. LastIn light of our comprehensive retrospective single-center analysis, the factors influencing the immune response to SARS-CoV-2 mRNA vaccination in recipients of allogeneic HSCT extend beyond simple post-transplantation timelines. A myriad of aspects, including concurrent immunosuppression therapies, particularly the use of MMF, CD19-positive B-cell counts, and even the choice of vaccine administered, play pivotal roles in determining humoral response efficacy. Notably, while a significant section of non-responders demonstrated a marked response to additional vaccine doses, the complexity of the immune response to SARS-CoV-2 cannot be captured by antibody response alone. T-cell responses, as recent research suggests, offer a robust reaction to vaccination even without detectable antibodies. This underlines the need for a holistic, multifaceted approach in tailoring vaccination strategies for allogeneic HSCT recipients. Careful consideration of immunosuppressant regimens, timely evaluations of B-cell counts, and perhaps even the nature of the vaccine itself may be crucial determinants. Nonetheless, while our findings provide significant insights and are supported by comparative studies, the retrospective nature and potential confounding factors necessitate cautious interpretation. Prospective, controlled studies will further elucidate and solidify the directions indicated by our research.